The Centres for Disease Control and Prevention (CDC) in United States classifies the biological agents that can possibly be used in a bioterrorist attack into three categories: A, B and C. According to the CDC, category A organisms are considered to pose the greatest risk to public safety. Category A organisms spread easily and the prevention of these agents requires rapid action. Furthermore, the protection against these agents requires large investments from the public health care. Category A agents consist of several viruses and three bacterial species: B. anthracis, Y. pestis and F. tularensis, which are the causative agents of anthrax, plague and tularemia, respectively.
The use of biological weapons does not necessarily appear instantly, since the incubation time of the organisms in the recipients can be several days, even weeks. It is important to develop diagnostic methods that allow a rapid diagnosis, because after the onset of clinical symptoms the clinical response to antimicrobial treatment is significantly reduced. Therefore, rapid and effective detection of exposure to these pathogens is essential for the initiation of prophylactic treatment before the onset of symptoms. In addition to antimicrobial treatment, prophylactic treatment includes e.g. immunization with vaccines.
The conventional methods for the detection of B. anthracis, Y. pestis and F. tularensis include the use of microbiological cultures and enzymatic, chemical and immunological assays. The classical microbiological diagnostic methods are often time-consuming and the cultivations can take several days. The need of additional enrichment tests may additionally slow down the procedure, as the pathogens must be distinguished from other, closely related species. Furthermore, the cultivation and enrichment of bacterial species may pose a health risk for laboratory personnel working in, e.g., a deployable field laboratory. Thus, rapid and accurate assays using molecular amplification technologies for the microbiological detection and identification of B. anthracis, Y. pestis and F. tularensis are essential to ensure proper medical intervention in the case of a suspected intentional release.
The most suitable microbiological tests for diagnosis of anthrax have been designed for blood culture, lumbar puncture or abscesses, in which case the samples are cultured for 1 day and subsequently further identified. In addition, fluorescent-antibody assays are available for rapid detection of anthrax and a result can be obtained in few hours. However, the result needs to be confirmed with alternative methods like PCR, because the specificity of these assays is not 100% reliable. WO 2004070001 discloses a hybridization based method for detecting B. anthracis nucleic acid in a sample. The method is based on two detection probes specific for genetic material contained on the pXO1 and pXO2 plasmids. The test is specific and reliable, however the hybridization technique used sets certain requirements for the laboratory work, especially if bacterial strains need to be cultured under the field conditions. Furthermore the analysis is time consuming.
Traditional methods for the identification of plague have several drawbacks. Y. pestis grows on general nutrient-rich media, but its growth rate is slower than that of most other bacteria. Therefore its presence may be masked by organisms that replicate faster. Furthermore, the characteristic bipolar staining of Y. pestis cells is not an exclusive feature limited to Y. pestis. Yersinia spp., enteric bacteria, and other gram-negative organisms, particularly Pasteurella spp., can exhibit the same characteristics in staining. Immunological tests for the diagnosis of Y. pestis may exhibit cross-reactivity, and therefore are not fully reliable.
F. tularensis may be identified by direct examination of secretions, exudates, or biopsy specimens using direct fluorescent antibody or immunohistochemical stains. F. tularensis demands a strict growth environment, thus making it hard to cultivate. F. tularensis is also one of the most infective bacteria known to man and the handling of infected samples presents an unnecessary risk to the personnel and Biosafety level 3 work protocols are advised. Antigen detection assays, PCR and enzyme-linked immunosorbent assay (ELISA) may be used to identify F. tularensis. Serum antibody titres do not attain diagnostic level until 10-14 days after onset of the illness. Therefore, serologic testing is only useful retrospectively: for definitive laboratory confirmation of the disease blood culture and increase in specific antibodies in paired sera are required. Accordingly, rapid identification of F. tularensis cannot be achieved with the presently available methods.
Polymerase chain reaction (PCR) analyses have been developed as alternatives for the classical microbiological methods. The traditional PCR is based on end-point analytics, wherein the amplified genetic material can only be inspected with a gel electrophoresis apparatus after the PCR reaction. A series of positive and negative PCR controls is needed to ensure the result and to eliminate the possibility of contamination during the PCR process.
A real-time PCR analysis with specific probes and primers designed for the target gene enables the detection of the PCR reactions already during the reaction. The real-time PCR offers several advantages for the analysis of biological agents that may be used in a bioterrorist attack. First, the real-time PCR is rapid, which is of primary importance in the case of both unintentional and deliberate release of such biological agents. Second, the realtime PCR suits for sensitive and specific pathogen detection, because it is performed in hermetically sealed wells, which greatly reduces the risk of cross-contamination, thereby diminishing the chance of false positive results. Third, the real-time PCR does not require post-PCR analysis.
Several studies disclose the use of a real-time PCR assay for the analysis of isolated bacterial organisms that can be used in a bioterrorist attack. Chase C. J. et al., 2005, Clin Chem 51 (10): 1778-1785, describe real time PCR assays targeting a unique chromosomal sequence of Y. pestis. The study indicates that by a real-time PCR assay it is possible to distinguish Y. pestis from other Yersinia species and from the closely related Y. pseudotuberculosis. Bell C. A. et al., 2002, J Clin Microbiol 40:2897-2902, discloses a rapid-cycle real-time PCR detection assay utilizing the LightCycler instrument (Roche Applied Science, Indianapolis) for cultured isolates of B. anthracis. Emanuel P. A. et al., 2003, J Clin Microbiol 41:689-693, disclose the detection of F. tularensis within infected mouse tissues by using a hand-held thermocycler and compare that to a real-time PCR analysis of tissue samples. However, none of these publications disclose a simultaneous detection of more than one pathogen species using a real-time PCR based method.
In spite of the recognized and obvious advantages of a simultaneous analysis of more than one bacterial pathogen that could be used as bioterrorism agents, no such analysis that would be specific and sensitive enough has so far been developed.
Varma Basil et al., 2004, Clin Chem 50 (6): 1060-1063, describe a real-time PCR assay that simultaneously detects four bacterial agents that could be used in bioterrorism. This study is based on molecular beacons that bind to amplicons generated from F. tularensis, Burkholderia mallei, Y. pestis and B. anthracis. The analysis takes advantage of 16S rRNA gene sequences, which are highly conserved among bacteria. With the described assay a simultaneous detection of the four pathogens was possible. However, the Y. pestis assay used cross-reacted strongly with 4 control bacterial species present in the samples. Such an assay would not be acceptable, since false-positive results in the diagnosis of category A agents would lead to significant economical losses and compromise the trust of the general public to the authorities.
Since the detection of Category A agents has to be absolutely accurate, rapid and reliable, further methods for efficient microbial detection of category A agents are still needed.